A critical step in regulating innate immune responses is the elaboration of pro-inflammatory factors such as TNF. TNF activates NF-kB and JNK signaling pathways, which induce the expression of other genes in a pro-inflammatory cascade. While it is clear that deregulated expression of TNF can cause inflammation, it is less clear how TNF signals are normally controlled in vivo. To better understand the mechanisms by which TNF signals are regulated and how such processes affect inflammation, we are studying A20, a novel molecule thought to regulate cellular responses to TNF. We have found that A20 is broadly expressed in multiple cell types, including both adaptive and innate immune cells, as well as nonhematopoietic cells. We have generated A20 deficient (A20-/-) mice, and these mice develop severe spontaneous inflammation and premature death. Preliminary cellular and genetic studies suggest that innate immune cells are severely affected by A20 deficiency in a cell-autonomous fashion. In addition, A20-/- cells display multiple defects in regulating TNF signals, indicating that A20 may be the first molecule identified to be essential for the termination of both TNF induced NF-kB activity and JNK activity. These observations highlight A20's pivotal roles in terminating TNF responses in vivo. It also provides a unique opportunity to interrogate the cellular and molecular mechanisms by which such control is normally achieved. Thus, we propose to: (1) determine the roles of A20 in regulating the homeostasis and function of innate immune cells; (2) determine the molecular mechanism by which A20 regulates NF-kB activity, and (3) determine the molecular mechanisms by which A20 regulates JNK activity. Understanding both the cellular and molecular mechanisms by which A20 regulates TNF responses will provide unique insights into how inflammatory responses are normally terminated, and how A20 regulates cellular activation and proliferation in vivo.